1. Field of the Invention
The present invention relates to a method of concentrating spots which are developed in a plate or paper, generically called a thin-layer chromatography (TLC) plate, for thin-layer chromatography or paper chromatography, and a measuring method for measuring spots which are developed in a TLC plate and sample components which are eluted through liquid chromatography (LC) with an infrared spectrometer such as a Fourier transform infrared (FTIR) spectrometer.
2. Description of the Background Art
A TLC plate for thin-layer chromatography is generally formed by a plate which is prepared by applying silica gel onto a support material such as a glass substrate or an aluminum sheet, while a filter paper is employed for paper chromatography.
FIG. 9 is a sectional view showing a conventional TLC plate for thin-layer chromatography. A silica gel layer 4 is formed on the surface of a glass substrate or aluminum sheet 2. Samples are developed in an in-plane direction of the silica gel layer 4 by an eluant, to define sample spots 6.
The sample spots thus developed in the TLC plate are directly measured with a densitometer, or scraped, extracted and concentrated to be thereafter subjected to spectrometry. However, the method of scraping, extracting and concentrating the spots is incorrect and troublesome.
There has been made an attempt to analyze components which are developed in a TLC plate with an FTIR spectrometer. In order to make analysis with the FTIR spectrometer, there have been proposed a method of directly applying focused infrared light to as-developed spots for measuring diffuse reflectance spectra (refer to Anal. Chem. 1989, 61, pp. 615-618), a method of scraping, extracting, and dripping spot portions on powder pellets of KBr or KCl and drying the same for thereafter measuring the same with an FTIR spectrometer (refer to Anal. Chem. 1987, 59, pp. 415-418), and a method of placing powder having no infrared absorption on an end portion of a TLC plate for transferring spot components from the TLC plate to the powder through glass fibers (refer to Anal. Chem. 1986, 58, pp. 2708-2714).
However, the method of directly applying infrared light to the TLC plate for FTIR measurement is inferior in practicability since silica gel contained in the TLC plate or the paper has large infrared absorption. On the other hand, the method of scraping, extracting, and dripping the component spots on the power pellets of KBr or KCl for FTIR measurement is incorrect and troublesome. In the method of transferring the spot components on the end portion of the TLC plate through the glass fibers, further, resolution in direction of development depends on the pitches of the glass fibers which are provided on the end portion, while this method is applicable only to a one-dimensionally developed TLC plate.
If infrared spectra of components separated through liquid chromatography can be obtained, it is extremely effective as qualitative means. In general, however, an eluant has strong absorption in an infrared region. Thus, it is impracticable to directly measure infrared spectra from an effluent in a flow cell system.
In order to obtain infrared spectra from an effluent in liquid chromatography, it is necessary to remove an eluant from the effluent. Various methods have been proposed for removing such eluants. One of these methods is adapted to drip an effluent on a powder layer of an infrared-nonabsorbent substance, such as KBr or KCl, which is reflective of infrared light, and to vaporize the eluant, thereby performing measurement with an infrared spectrometer. However, this method is impracticable since the powder of the infrared-nonabsorbent substance, such as KBr or KCl, is easily dissolved in water which is contained in the eluant.
Other methods are adapted to drip an effluent on a metal belt, and to spray an effluent onto a metal belt for facilitating vaporization of the eluant. However, these methods are also impracticable due to problems of spreading of spots and low detection sensitivity.